Dinoflagellates are marine unicellular eukaryotes that exhibit unique features including a very low level of basic proteins bound to the chromatin and the complete absence of histones and nucleosomal structure. A cDNA encoding a protein with a strong homology to the TATA box-binding proteins (TBP) has been isolated from an expressed sequence tag library of the dinoflagellateCrypthecodinium cohnii. The typical TBP repeat signature and the amino acid motives involved in TFIIA and TFIIB interactions were conserved in this new TBP-like protein. However, the four phenylalanines known to interact with the TATA box were substituted with hydrophilic residues (His77, Arg94, Tyr171, Thr188) as has been described for TBP-like factors (TLF)/TBP-related proteins (TRP). A phylogenetic analysis showed that cTBP is intermediate between TBP and TLF/TRP protein families, and the structural similarity of cTBP with TLF was confirmed by low affinity binding to a consensus' TATA box in an equivalent manner to that usually observed for TLFs. Six 5′-upstream gene regions of dinoflagellate genes have been analyzed and neither a TATA box nor a consensus-promoting element could be found within these different sequences. Our results showed that cTBP could bind stronger to a TTTT box sequence than to the canonical TATA box, especially at high salt concentration. Same binding results were obtained with a mutated cTBP (mcTBP), in which the four phenylalanines were restored. To our knowledge, this is the first description of a TBP-like protein in a unicellular organism, which also appears as the major form of TBP present in C. cohnii. Dinoflagellates are marine unicellular eukaryotes that exhibit unique features including a very low level of basic proteins bound to the chromatin and the complete absence of histones and nucleosomal structure. A cDNA encoding a protein with a strong homology to the TATA box-binding proteins (TBP) has been isolated from an expressed sequence tag library of the dinoflagellateCrypthecodinium cohnii. The typical TBP repeat signature and the amino acid motives involved in TFIIA and TFIIB interactions were conserved in this new TBP-like protein. However, the four phenylalanines known to interact with the TATA box were substituted with hydrophilic residues (His77, Arg94, Tyr171, Thr188) as has been described for TBP-like factors (TLF)/TBP-related proteins (TRP). A phylogenetic analysis showed that cTBP is intermediate between TBP and TLF/TRP protein families, and the structural similarity of cTBP with TLF was confirmed by low affinity binding to a consensus' TATA box in an equivalent manner to that usually observed for TLFs. Six 5′-upstream gene regions of dinoflagellate genes have been analyzed and neither a TATA box nor a consensus-promoting element could be found within these different sequences. Our results showed that cTBP could bind stronger to a TTTT box sequence than to the canonical TATA box, especially at high salt concentration. Same binding results were obtained with a mutated cTBP (mcTBP), in which the four phenylalanines were restored. To our knowledge, this is the first description of a TBP-like protein in a unicellular organism, which also appears as the major form of TBP present in C. cohnii. In higher eukaryotes, the regulation of transcription is intimately connected to the chromatin structure, and the accessibility of the transcription factors to their recognition elements is facilitated by the chromatin-remodeling processes involving a subset of modifying machines whose properties can alter the nucleosomal structure (1Armstrong J.A. Emerson B.M. Curr. Op. Genet. Dev. 1998; 8: 165-172Google Scholar, 2Cairns B.R. Trends Biochem. Sci. 1998; 23: 20-25Google Scholar, 3Cox J.M. Kays A.R. Sanchez J.F. Schepartz A. Curr. Biol. 1998; 2: 11-17Google Scholar, 4Gregory P.D. Hörz W. Curr. Op. Cell Biol. 1998; 10: 339-345Google Scholar, 5Kadonaga J.T. Cell. 1998; 92: 307-313Google Scholar, 6McAdams H.H. Arkin A. Curr. Biol. 2000; 10: R318-R320Google Scholar, 7Uhlmann F. Curr. Biol. 2001; 11: R384-R387Google Scholar, 8Wolffe A.P. Hayes J.J. Nucleic Acids Res. 1999; 27: 711-720Google Scholar, 9Workman J.L. Kingston R.E. Annual Rev. Biochem. 1998; 67: 545-579Google Scholar, 10Wu C. J. Biol. Chem. 1997; 272: 28171-28174Google Scholar). After the relief of repression of the chromatin, transcription is preinitiated by the interaction of the RNA polymerase and the general transcription factors with the promoter. In mRNA synthesis, the transcription initiation step begins with the recognition of the promoter by the TFIID complex containing the TATA box-binding protein (TBP) 1The abbreviations used are: TBP, TATA box-binding protein; TLF, TBP-like factor; TRP, TBP-related protein; TRP, TBP-related protein; PCP, peridinin chlorophyll-a-binding protein; RACE, rapid amplification of cDNA ends; GST, glutathioneS-transferase; AdMLP, adenovirus major late promoter; EST, expressed sequence tag; TF, transcription factor; cTBP, C. cohnii TBP; hsTBP, h. sapiens TBP. and several TBP-associated factors (11Berk A.J. Curr. Op. Cell Biol. 1999; 11: 330-335Google Scholar, 12Buratowski S. Curr. Op. Cell Biol. 2000; 12: 320-325Google Scholar, 13Klug A. Science. 2001; 292: 1844-1846Google Scholar, 14Ranish J.A. Hahn S. Curr. Op. Genet. Dev. 1996; 6: 151-158Google Scholar, 15Johnson K.M. Mitsouras K. Carey M. Curr. Biol. 2001; 11: R510-R513Google Scholar). The TBP, which is highly conserved among eukaryotes, was considered until recently as the universal transcription initiator factor (16Bareket-Samish A. Cohen I. Haran T.E. J. Mol. Biol. 2000; 299: 965-977Google Scholar, 17Burley S.K. Curr. Op. Struct. Biol. 1996; 6: 69-75Google Scholar, 18Rowlands T. Bauman P. Jackson S.P. Science. 1994; 264: 1326-1329Google Scholar). However, new members of the TBP family called TBP-like factors (TLF) or TBP-related proteins (TRP) were identified only in the metazoan. Many studies showed that these new factors could form a stable complex with TFIIA and TFIIB and substitute for TBP in directing transcription in vitro by RNA polymerase II (reviewed in Refs. 19Dantonel J.C. Wurtz J.M. Poch O. Moras D. Tora L. Trends Biochem. Sci. 1999; 24: 335-339Google Scholar, 20Berk A.J. Cell. 2000; 103: 5-8Google Scholar). In higher eukaryotes, promoters do not always contain a TATA box but show an initiator element, which is loosely conserved and encompasses the transcription start site (21Pugh B.F. Tjian R. Genes Dev. 1991; 5: 1935-1945Google Scholar, 22Patikoglou G.A. Kim J.L. Sun L. Yang S.-H. Kodadek T. Burley S.K. Genes Dev. 1999; 13: 3217-3230Google Scholar). In protists, the TATA box is found in amoebas (Acanthamoeba), in slime mold (Dictyostelium), in ciliates (Histriculus cavicola), and in apicomplexa (Plasmodium) (23Wong J.M. Liu F. Bateman E. Nucleic Acids Res. 1992; 20: 4817-4824Google Scholar, 24Huang W. Bateman E. J. Biol. Chem. 1995; 270: 28839-28847Google Scholar, 25Cohen S.M. Knecht D. Lodish H.F. Loomis W.F. EMBO J. 1986; 5: 3361-3366Google Scholar, 26Kimmel A.R. Firtel R.A. Nucleic Acids Res. 1983; 11: 541-552Google Scholar, 27Liston D.R. Johnson P.J. Mol. Cell. Biol. 1999; 19: 2380-2388Google Scholar, 28McAndrew M.B. Read M. Sims P.F.G. Hyde J.E. Gene. 1993; 124: 165-171Google Scholar). In trypanosomatida (Kinetoplastidae) and trichomonadida (Parabasalia), neither the TATA box nor analogous sequences were detected among the few characterized genes. However, both showed an initiator element specific to each phylum (29Luo H. Gilinger G. Mukherjee D. Bellofatto V. J. Biol. Chem. 1999; 274: 31947-31954Google Scholar, 30Quon D.V.K. Delgadillo M.G. Johnson P.J. J. Mol. Evol. 1996; 43: 253-262Google Scholar, 31Quon D.V.K. Delgadillo M.G. Khachi A. Smale S.T. Johnson P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4579-4583Google Scholar). Dinoflagellates are protists, which are widely distributed in the aquatic environment. These unicellular microorganisms can be free living or parasitic. Both toxic and non-toxic dinoflagellates can proliferate in seawater, causing important economic and health problems. The most prominent feature of dinoflagellate cell biology, unique among eukaryotic cells, is the lack of histones and nucleosomal organization (32Soyer-Gobillard M.O. Moreau H. Encyclopedia of Microbiology. Academic Press, Orlando, FL2000: 42-54Google Scholar, 33Raikov I.B. Acta Protozool. 1995; 34: 239-247Google Scholar, 34Spector D.L. Dinoflagellates. Academic Press, Orlando, FL1984: 1-15Google Scholar, 35Herzog M. Soyer M.O. Eur. J. Cell Biol. 1981; 23: 295-302Google Scholar, 36Herzog M., De Marcillac G.D. Soyer M.O. Eur. J. Cell Biol. 1982; 27: 151-155Google Scholar). Moreover, conversely to other eukaryotes, the dinoflagellate chromosomes remain highly condensed during the G1 phase, with DNA filaments protruding from the chromosome core where transcription takes place (37Bhaud Y. Guillebault D. Lennon J.F. Defacque H. Soyer-Gobillard M.O. Moreau H. J. Cell Sci. 2000; 113: 1231-1239Google Scholar). The upstream gene organization is only known in the dinoflagellate speciesGonyaulax polyedra for two genes: the peridinin chlorophyll-a-binding protein (PCP) and the luciferase genes (38Le Q.H. Markovic P. Hastings J.W. Jovine R.V.M. Morse D. Mol. Gen. Genet. 1997; 255: 595-604Google Scholar, 39Li L. Hastings J.W. Plant Mol. Biol. 1998; 36: 275-284Google Scholar). These two genes exhibited a tandem repeat spaced by an intergenic region of about 1000 bp that contains a common 13-bp sequence, but no TATA box or other known regulatory elements have been found. To date, only two proteins involved in transcription have been described in dinoflagellate (40Guillebault D. Derelle E. Bhaud Y. Moreau H. Protist. 2001; 152: 127-138Google Scholar). The elucidation of their transcription machinery could allow these organisms to be used as powerful models for the study of eukaryotic transcription in an environment devoid of nucleosomes and provide a better understanding of the transcription network in other eukaryotes. In this work, we identified for the first time in a unicellular organism a cDNA encoding a novel TBP-like protein containing mutated key amino acids involved in DNA binding. We also analyzed the 5′-upstream part of four genes of the dinoflagellate Crypthecodinium cohnii and of two genes of the dinoflagellate Gonyaulax polyedra without any evidence of any known regulatory elements. We compared the binding of the cTBP and of a mutated form (mcTBP) to a TTTT box and to a canonical TATA box in various salt concentrations. The TBP cDNA was inserted into a pBlueScript vector and was amplified by the polymerase chain reaction using the NdeI restriction site containing primer 5′-TCA CAA TGT CAT ATG GCG GAT ATC TTG GAA-3′ and theXhoI restriction site containing primer 5′-TAG ATT ATA CTC GAG GGT CTT GAA CTC CGC-3′. The PCR product was subcloned into the pGEX4T expression vector (Amersham Biosciences). The fusion protein GST-cTBP was produced in the Escherichia coli strain after 1 mmisopropyl-1-thio-β-d-galactopyranoside induction and purified using glutathione-Sepharose beads (Amersham Biosciences) as described elsewhere (41Moore P.A. Ozer J. Salunek M. Jan G. Zerby D. Campbell S. Lieberman P.M. Mol. Cell. Biol. 1999; 19: 7610-7620Google Scholar). The clones producing recombinant proteins were sequenced to check the absence of mutation. The C. cohniicDNA λ zap expression library was plated in N-Z-amine yeast extract medium Top agar media on NZY agar plates and incubated overnight at 37 °C. Plates were covered by nitrocellulose for The were for in for in and for in After for 1 at the were for 1 at in a containing and 1 DNA and were in the with a cTBP overnight at After with the and the nitrocellulose were and to for h. was until the clones were were from the and the were were with F. Poch O. 2000; using K. F. Nucleic Acids Res. 1997; and the with 1993; 6: Scholar). The phylogenetic was using the with the M. C. 1996; 12: Scholar). The 5′-upstream sequences of and cohnii genes were amplified by using the universal and specific at the of the (40Guillebault D. Derelle E. Bhaud Y. Moreau H. Protist. 2001; 152: 127-138Google Scholar, J. Soyer-Gobillard M.O. Bhaud Y. I. T. Moreau H. Protist. 2000; Scholar). After and of the amplified DNA regions were used to with the were with purified fusion proteins and The adenovirus major late promoter sequence from to to the start or the mutated sequence in which the box is substituted with was by of the using DNA binding were with of as or of GST-cTBP or or of TBP were for at with or of in a containing 1 and was and incubated for a at °C. and TFIIA were purified as described in Refs. C. S. I. J. J.M. Tora L. P. EMBO J. 1993; 12: and A. Y. D. A. I. I. P. Tora L. EMBO J. 1994; 13: The study of the salt on the DNA binding of GST-cTBP was in the but with an in the from to The were on a at in at for the The was and to The cTBP cDNA in pBlueScript vector was mutated to a at the residues Arg94, Tyr171, and by PCR using the four of for each of the four The cDNA was amplified The PCR were overnight at and in the E. The and were by A C. cohnii library was analyzed and an to the TBP family was identified using the The cDNA was sequenced and showed an of bp encoding for a protein. The and that this novel protein showed the typical signature of TBP the first amino acids of the showed with the region and The two each amino acids in which is the typical TBP The between these two was to that in of other organisms yeast The region of the cTBP amino no homology as is usually described in other eukaryotic key amino acids known to be involved in with TFIIA and were also conserved (41Moore P.A. Ozer J. Salunek M. Jan G. Zerby D. Campbell S. Lieberman P.M. Mol. Cell. Biol. 1999; 19: 7610-7620Google Scholar). The most between cTBP and the was the of the two of the highly conserved which are known to a key in the DNA by by the and in the first and the repeat of cTBP a amino acid was also observed in the TLF feature could in the recognition of a DNA element different from a TATA box J.C. Wurtz J.M. Poch O. Moras D. Tora L. Trends Biochem. Sci. 1999; 24: 335-339Google Scholar). the sequence cTBP to the similarity with than to the with the interaction between TBP and the transcription factors TFIIA and TFIIB were highly conserved both in cTBP and Hahn S. S. 1996; 272: Scholar, H. K. Burley S.K. 1995; Scholar). these that cTBP was the to than to any TBP-like protein identified to to TBP members was also by phylogenetic analysis where cTBP in a in the TBP and was from the TLF as by In this cTBP as a of a new family of transcription which be in the TBP or cTBP was isolated after of an The that a canonical TBP could be To that this new cTBP was not a form of TBP, from a λ cDNA library of C. cohnii were at low using a the first repeat of the cTBP To check the were for the of TBP as as TLF or TRP, a of the yeast DNA was as contains only a TBP gene A.J. Cell. 2000; 103: 5-8Google Scholar). A was that the the of TBP from the C. cohnii cDNA Six clones were and after to the cTBP sequence, including the substituted residues that be involved in DNA binding. These results that the identified cTBP was the major form of a TBP family in C. cohnii. The of TBP, TLF, and sequences in 1 is a subset of a sequences with and with not F. Poch O. 2000; Scholar, K. F. Nucleic Acids Res. 1997; Scholar, 1993; 6: Scholar). the low sequence with the TBP cTBP exhibited a few amino acid and a homology has been the TBP structure as a J.C. Wurtz J.M. Poch O. Moras D. Tora L. Trends Biochem. Sci. 1999; 24: 335-339Google Scholar, H. K. Burley S.K. 1995; using the A. J. Mol. Biol. 1993; Scholar). The residues in the and of cTBP and were conserved These especially and are found in eukaryotic and are to a structure a between and in each In a few other residues were highly conserved at the as in the other both in the and repeat of cTBP These residues to the core of the and form a core in each repeat a conserved salt between residues and for the which the two cTBP two amino acids and which a However, the structure of cTBP, by the network of C. R. 1994; 10: the organization as the for the TBP structure. these that cTBP most a structure to TBP major amino acid In the first the two residues and in are by a and an in cTBP and which with and form a network and A of interaction has been observed in the repeat of TLF with the amino which in the structure J.C. Wurtz J.M. Poch O. Moras D. Tora L. Trends Biochem. Sci. 1999; 24: 335-339Google Scholar). In the the residues and are by and and to the by the a few other a that be to the with DNA major within the the present in of the of a complex to the observed in the box structure. However, the DNA by this novel of interactions that the DNA element by cTBP be different from the TATA box as has been for TLFs. The in C. cohnii of a major TBP factor at the key amino acids involved in the TATA box binding to study the structure of the promoter region of new genes in this We amplified and sequenced the region of four new genes by of these genes the highly expressed protein and the proteins and and (40Guillebault D. Derelle E. Bhaud Y. Moreau H. Protist. 2001; 152: 127-138Google J. Soyer-Gobillard M.O. Bhaud Y. I. T. Moreau H. Protist. 2000; Scholar). The upstream sequences were with of the and luciferase genes from the dinoflagellate a TATA box nor any other known promoter element could be found within the first 1000 upstream of the start not for the two dinoflagellate upstream sequences of the and the luciferase known in G. where no TATA box nor any promoter element could be identified (38Le Q.H. Markovic P. Hastings J.W. Jovine R.V.M. Morse D. Mol. Gen. Genet. 1997; 255: 595-604Google Scholar, 39Li L. Hastings J.W. Plant Mol. Biol. 1998; 36: 275-284Google Scholar). The transcription initiation site has been identified in the luciferase sequences could not be found in the promoters of the genes identified L. Hastings J.W. Plant Mol. Biol. 1998; 36: 275-284Google Scholar). cTBP was produced as a protein in E. coli To study in DNA a protein (mcTBP), in which the four amino acids known to to the of the four phenylalanines involved in the DNA binding were by was also produced The and the TBP were incubated with the or mutated and to A of the TATA was observed with the only a very low binding was obtained in the of a of cTBP The of TFIIA in the not the of the complex The observed by the cTBP with the mutated TATA was stronger compared with the by the incubated with the the also bound to the mutated TATA for the TATA and for the mutated and in general showed a binding to the However, in the of an in the binding to the canonical TATA box by was observed with the tag and the TFIIA were to that no binding of these to the DNA was obtained A Moreover, as described not bind to the mutated TATA box, in the of TFIIA and cTBP is characterized by amino acid residues in the DNA binding we a high salt could DNA as for R. J.E. J. Mol. Biol. 1998; Scholar). in the binding of the cTBP to the TATA box with the with an However, the high not the cTBP binding In a to was at low salt the binding was important on the mutated than on the canonical TATA element not In this we for the first time in a unicellular eukaryotic organism a new of transcription initiation factors that show intermediate structural features between the TBP and TLF/TRP family of However, our DNA binding results that this novel protein proteins than cTBP not bind to the TATA Dinoflagellates are eukaryotes the unique feature of a very low level of basic proteins to their chromatin and a complete absence of nucleosomal structure (32Soyer-Gobillard M.O. Moreau H. Encyclopedia of Microbiology. Academic Press, Orlando, FL2000: 42-54Google Scholar, P.J. J. Protozool. 1991; Scholar). is known about the processes of dinoflagellate and a RNA polymerase II has been described in the C. the has not been isolated P.J. J. Protozool. Scholar). The chromosomes are highly condensed during the and has been that transcription at the of the chromosomes (37Bhaud Y. Guillebault D. Lennon J.F. Defacque H. Soyer-Gobillard M.O. Moreau H. J. Cell Sci. 2000; 113: 1231-1239Google Scholar, Scholar). proteins were isolated and characterized their in transcription cohnii and the cTBP is the first dinoflagellate (40Guillebault D. Derelle E. Bhaud Y. Moreau H. Protist. 2001; 152: 127-138Google Scholar, Y. J. Soyer-Gobillard M.O. Moreau H. J. 1998; Scholar). The of the upstream sequence of four C. cohnii genes confirmed the absence of a TATA element as described in two genes of dinoflagellate The dinoflagellate promoter gene sequences showed a high in their for each of the four and no transcription initiation was found from the sequence A 13-bp sequence identified in the two G. polyedra genes was not found in the new sequences. 13-bp sequence is specific to G. polyedra or to the highly expressed and luciferase genes or is not a transcription In the luciferase gene this 13-bp sequence is about bp upstream of the transcription initiation from the for the TATA element (38Le Q.H. Markovic P. Hastings J.W. Jovine R.V.M. Morse D. Mol. Gen. Genet. 1997; 255: 595-604Google Scholar, 39Li L. Hastings J.W. Plant Mol. Biol. 1998; 36: 275-284Google Scholar). of cTBP with and a structure described in proteins to the TBP family and also the in the DNA sequence These with our results in which the low binding of cTBP to the TATA box in DNA binding that is to a TLF/TRP S. J.T. R. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar, Y. T. Nucleic Acids Res. 1999; 27: Scholar). A low binding to the TATA box was observed for the and no sequence by these proteins is known J.C. Wurtz J.M. Poch O. Moras D. Tora L. Trends Biochem. Sci. 1999; 24: 335-339Google Scholar). The of the of salt on the cTBP interaction with DNA a where DNA binding be by on the DNA sequences to be in a highly condensed environment. is known about the of the four phenylalanines the TATA box binding. be that the of the phenylalanines the to bind the TATA box but this was not can be by a structure of the cTBP in which the could a the protein to bind However, in the of the cTBP containing the four showed a binding to the TATA box, that the four phenylalanines be involved in the TATA box binding The of the TLF/TRP proteins in a few that the initiation of transcription was complex than These proteins are to be on genes involved in specific in several organisms S. L. M. Tora L. Mol. Cell. 2000; 6: Scholar, Tjian R. Science. 2000; Scholar, L. Mol. Cell. 2000; 6: Scholar, D.L. A.P. Science. 2000; Scholar, F. L. U. Tora L. Curr. Biol. 2001; 11: Scholar). have only been in and not in unicellular in S. for which the is sequenced and J.C. Wurtz J.M. Poch O. Moras D. Tora L. Trends Biochem. Sci. 1999; 24: 335-339Google Scholar, 20Berk A.J. Cell. 2000; 103: 5-8Google Scholar). The expression of the cTBP as the major TBP-related protein in the unicellular organism C. which not have that to transcription can the as the TBP found in the could different initiation sequences that different in other is to a between the unique structure of dinoflagellate chromatin, the absence of TATA or any upstream element, and the of the cTBP as the major TBP protein (32Soyer-Gobillard M.O. Moreau H. Encyclopedia of Microbiology. Academic Press, Orlando, FL2000: 42-54Google Scholar). for the of unique transcription initiation factors in other dinoflagellate in other unicellular eukaryotes be to study this and We M. M. and C. for and and for the We for of the We are to for and E. and M. for at a of this
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